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Evolutionary-developmental study of membrane proteins
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Baluška, František (referee) ; Štorchová, Helena (referee)
Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...
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Analysis of CESA complexes dynamics in plant cytoskeletal mutants
Dubenecká, Kamila ; Schwarzerová, Kateřina (advisor) ; Malínská, Kateřina (referee)
The basis of this study are mutant plants with ARP2/3 complex lacking in one of its subunits (arpc5 and arp2). These plants also express CSC subunit CESA6 of primary cell wall tagged by YFP. Thanks to modern imaging technologies, it is possible to observe the movement of tagged cellulose synthase complexes in vivo at plasmatic membrane. Kymograph analyses was used to measure the velocity of CESA complexes. In addition to observing CESA complexes directly on the plasma membrane, experiments were made to regenerate cell walls of protoplasts of Arabidopsis thaliana plants arpc5 and WT. It was found, that observed mutants arpc5 and arp2 have reduced velocity of CESA complexes in comparison to WT and arpc5 protoplasts regenerate cellulose mesh of cell wall slower. Keywords: Cellulose synthesis, ARP2/3 complex, CESA, CSC velocity, arpc5, arp2, Arabidopsis thaliana.
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Endocytosis in plants
Svitáková, Lenka ; Krtková, Jana (advisor) ; Hála, Michal (referee)
Endocytosis is a process typical for eukaryotic cells. It is a mechanism that ensures invagination of the plasma membrane which leads to the creation of a membrane vesicle with specifically selected cargo. The vesicle is then internalized by the cell. Endocytosis has a crucial role in plant growth and development not only thanks to the reception of nutrients from the extracellular space but also by having a huge impact on the membrane composition which influences cellular signalling. There are few types of endocytosis known in plants. They differ from each other by the molecular machinery which ensures their process. The most studied type in plants is the clathrin-mediated endocytosis. Clathrin-coated vesicle formation proceeds through five stages. However, there are less details known about it in plant cells than in animal cells. In the first part of my bachelor thesis I describe recent knowledge of this topic and the most important participants in this process. I also mention the mechanisms of regulations that plant cells use to coordinate clathrin-mediated endocytosis. In the second part I summarize recently discovered types of endocytosis in plants that are independent from clathrin. These are endocytosis associated with microdomains and fluid- phase endocytosis. The details of these endocytic...
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The influence of aluminum toxicity on the dynamics of plant cortical microtubules
Pohl, Jana ; Schwarzerová, Kateřina (advisor) ; Pejchar, Přemysl (referee)
Aluminium toxicity is the main factor limiting plant growth on acid soils. Aluminium inhibits root growth within few minutes after aluminium treatment. The mechanism and primary target of his action is still unknown. In this diploma thesis the effect of aluminium toxicity on dynamics of cortical microtubules WT and pldα1 plants was studied using the EB1a-GFP marker. Polymerization rate in both the transition and the elongation zone increased immediately after the aplication of aluminium. Nevertheless, microtubules in the transition zone are much more sensitive to aluminium, because the aluminium-induced increase in the polymerization rate was higher than in the elongation zone. Plants lacking PLDα1 showed higher dynamics on plus ends of cortical microtubules compared to WT during aluminium stress, which enabled them to react faster to stress stimuli. Mutants showed lower sensitivity to aluminium and 100 μM concentration of aluminium ions has beneficial effect on root growth in pldα1. These results suggest that PLDα1 influences microtubule dynamics. Microtubules in pldα1 plants were more dynamic and they polymerized faster in the response to aluminium, which was accompanied by decreased sensitivity to aluminium stress compared to WT. Changes in microtubule dynamics may play a role in aluminium...
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Role of protein NtRGS1 in cell signaling and regulation of growth of tobacco BY-2 cell line.
Šonka, Josef ; Srba, Miroslav (advisor) ; Martinec, Jan (referee)
5 Abstract The thesis is focused on the role of regulator of G-protein signaling NtRGS1 in control of growth and cell proliferation of tobacco cell line BY-2. The protein NtRGS1 is an important candidate for being plant G-protein coupled receptor. Heterotrimeric G-proteins are involved in key signaling mechanisms in eukaryotic cells. Basic principles of this type of signaling are well conserved between plants and animals and related higher taxa. Outstanding difference of plant G-protein system is altered enzymatic activity of Gα subunit of the G-protein heterotrimer. These alterations correlate with chimeric structure and function of investigated NtRGS1 protein. The interaction of Gα and NtRGS1 is absolutely essential for running of heterotrimeric G-protein signaling in plants. Truncated versions of NtRGS1 fused to GFP were crated in the aim of protein characterization. The truncated proteins were investigated in respect of analysis of the role of NtRGS1 domains in protein targeting. Dynamic changes in NtRGS1 and selected truncated versions induced by experimental application of nutrition, especially sugars were described. Expression if Gα and NtRGS1 were investigated simultaneously. Influence of modulation of Gα and NtRGS1 expression on growth parameters of tobacco cell line BY-2 were described. Key words:...
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The role of ARP2/3 complex in plant cells
Schiebertová, Petra ; Schwarzerová, Kateřina (advisor) ; Martinec, Jan (referee)
2 Abstract ARP2/3 protein complex is formed from seven proteins (ARP2, ARP3 and ARC1- ARPC5) with a relatively conserved structure. ARP2/3 complex branches and nucleates new actin filaments. This thesis focuses on the study of the role and importance of the individual subunits of the complex ARP2/3 in plants. One of the principal aims of this work is to determine whether complex ARP2/3 may at least partially maintain its role when one or more of the subunits are not available. Furthermore if the individual subunits play another, plant-specific role and if the subunits are functionally equivalent in the complex. The main way how to achieve this objective is the analysis of multiple mutants of Arabidopsis thaliana in subunits of ARP2/3 complex. After comparing several phenotypes of mutant lines it is obvious that all the subunits are functionally equivalent. A loss of ARPC5 subunit usually manifests the strongest phenotypic expression. On the contrary, loss ARPC3 and ARPC2b subunits have weak phenotypic manifestations. Because some phenotypes, such as phenotype distorted trichomes was detected only in some mutant lines, whereas the phenotype of faster roots gravitropic response or vacuolar system fragmentation that was detected in all analyzed mutants suggests, that different subunits play varying roles...
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The role of ARPC2 in plant cells
Šlajcherová, Kateřina ; Schwarzerová, Kateřina (advisor) ; Klíma, Petr (referee)
ARPC2 protein localization in a plant cell Kateřina Šlajcherová 1 Abstract Actin cytoskeleton is an ubiquitous structure which plays numerous irreplacable roles. Actin nucleation is, beside formins, performed by ARP2/3 complex (actin-related protein), comprising of seven subunits (ARP2, 3, C1-C5) and activated by protein SCAR/WAVE complex. ARP2/3 complex is attached to the membrane and branches existing microfilaments, apart from nucleating them de novo. ARP2/3 mutants in most organisms show severe defects. However, plant mutants exhibit only mild phenotype, for example, Arabidopsis thaliana ARPC2 mutant (dis2-1) has deformed trichomes and leaf epidermal cells, but its viability is not impaired. The aim of the thesis is to map ARPC2 localization within the cell and broaden our understanding of ARP2/3 complex role in plant cell morphogenesis. Tobacco ARPC2 (NtArpC2) subunit was visualized in Arabidopsis plants, using the GFP fusion protein as well as imunofluorescence and anti-ARPC2 antibody. Experiments were undertaken to collocalize the subunit with actin and microtubular cytoskeleton, with mitochondrions, endosomes and other membrane organelles. The specimens were observed in confocal and TIRF microscope. The GFP-NtARPC2 protein shows as motile dots; their movement, but not their existence, is dependent...
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Microtubule-associated proteins in plants
Benáková, Martina ; Krtková, Jana (advisor) ; Vinopal, Stanislav (referee)
1. Abstract and key words MTs are one of the basic cellular protein structure. Their features and function are influenced and modified by group of other proteins, i.e. microtubule-associated proteins (MAPs). In the last decades, an extensive research on MAPs and their wide range of functions has been carried out. Therefore we are aware of the involvement of some of the MAPs in MT dynamics, other have been shown to have rather structural function. They bundle MTs with various cell structures, such as the other MTs, proteins, organelles, actin cytoskeleton or plasma membrane. Many described MAPs are homologous in the whole eukaryotic domain, for example MAP65 or EB1 (END BINDING 1) family, therefore it is interesting to follow if and how the functions of plant MAPs differ from their animal counterparts. On the other hand, there are many specific MAPs with unique functions in plants, e.g. ATK5 or SPR1 (SPIRAL 1). This Bachelor thesis is a survey on current knowledge of plant MAPs and it makes an effort to present their characteristic and functions in plant cell and organism. Key words: cytoskeleton, microtubules, microtubule-associated proteins, plant cell, growth and development
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